ANKRD1 aggravates renal ischaemia‒reperfusion injury via promoting TRIM25‐mediated ubiquitination of ACSL3

ABSTRACT Background Renal ischaemia‒reperfusion injury (IRI) is the primary cause of acute kidney injury (AKI). To date, effective therapies for delaying renal IRI and postponing patient survival remain absent. Ankyrin repeat domain 1 (ANKRD1) has been implicated in some pathophysiologic processes, but its role in renal IRI has not been explored. Methods The mouse model of IRI‐AKI and in vitro model were utilised to investigate the role of ANKRD1. Immunoprecipitation‐mass spectrometry was performed to identify potential ANKRD1‐interacting proteins. Protein‒protein interactions and protein ubiquitination were examined using immunoprecipitation and proximity ligation assay and immunoblotting, respectively. Cell viability, damage and lipid peroxidation were evaluated using biochemical and cellular techniques. Results First, we unveiled that ANKRD1 were significantly elevated in renal IRI models. Global knockdown of ANKRD1 in all cell types of mouse kidney by recombinant adeno‐associated virus (rAAV9)‐mitigated ischaemia/reperfusion‐induced renal damage and failure. Silencing ANKRD1 enhanced cell viability and alleviated cell damage in human renal proximal tubule cells exposed to hypoxia reoxygenation or hydrogen peroxide, while ANKRD1 overexpression had the opposite effect. Second, we discovered that ANKRD1's detrimental function during renal IRI involves promoting lipid peroxidation and ferroptosis by directly binding to and decreasing levels of acyl‐coenzyme A synthetase long‐chain family member 3 (ACSL3), a key protein in lipid metabolism. Furthermore, attenuating ACSL3 in vivo through pharmaceutical approach and in vitro via RNA interference mitigated the anti‐ferroptotic effect of ANKRD1 knockdown. Finally, we showed ANKRD1 facilitated post‐translational degradation of ACSL3 by modulating E3 ligase tripartite motif containing 25 (TRIM25) to catalyse K63‐linked ubiquitination of ACSL3, thereby amplifying lipid peroxidation and ferroptosis, exacerbating renal injury. Conclusions Our study revealed a previously unknown function of ANKRD1 in renal IRI. By driving ACSL3 ubiquitination and degradation, ANKRD1 aggravates ferroptosis and ultimately exacerbates IRI‐AKI, underlining ANKRD1's potential as a therapeutic target for kidney IRI. Key Points/Highlights Ankyrin repeat domain 1 (ANKRD1) is rapidly activated in renal ischaemia‒reperfusion injury (IRI) models in vivo and in vitro. ANKRD1 knockdown mitigates kidney damage and preserves renal function. Ferroptosis contributes to the deteriorating function of ANKRD1 in renal IRI. ANKRD1 promotes acyl‐coenzyme A synthetase long‐chain family member 3 (ACSL3) degradation via the ubiquitin‒proteasome pathway. The E3 ligase tripartite motif containing 25 (TRIM25) is responsible for ANKRD1‐mediated ubiquitination of ACSL3.


Fig. S3 .
Fig. S3.ANKRD1 expression is elevated in renal tubular epithelial cells in renal IRI.A and B. The scRNA-seq data revealed that the expression of ANKRD1 in proximal tubular epithelial cells (PT S3 and S1) of mouse kidneys significantly increased after experiencing short (23 min) or long (30 min) bilateral ischemia.scRNA-seq data are obtained from Susztaklab Kidney Biobank: Mouse Kidney IRI scRNA-seq (https://susztaklab.com/Mouse_IRI_scRNA/index.php).Experimental grouping: Male C57BL/6 mice were subjected to short and long ischemia and followed for 1, 3 and 14 d.Bulk and scRNA-seq was performed in n=2 mice per condition and time point and compared to n = 6 controls.C and D. Serum SCr and BUN indicated a significant decrease of renal function in renal I/R-treated mice.E. Quantitative analysis of renal tubular epithelial cell damage.F. Immunoblot analysis of ANKRD1, KIM-1 and NGAL in H2O2-treated HK-2 cells with β-actin as a loading control.***P < 0.001.

Fig. S4 .
Fig. S4.Knockdown of ANKRD1 in mice via tail vein attenuated IRI-induced kidney injury and ferroptosis.Alterations in the protein levels of KIM-1, ACSL3 and GPX4 were detected by Western blot after tail vein injection of rAAV9 to intervene with ANKRD1 levels in mice kidney.

Fig
Fig. S5.ANKRD1 exacerbates H/R-induced renal tubular epithelial cell injury and ferroptosis.A. Cell viability of HK-2 cells under different treatment conditions was analyzed by CCK-8.B. Representative cellular images of ANKRD1 and KIM-1 IF staining.Scale bar, 50 μm.

Fig. S9 .
Fig. S9.TRIM25 is implicates in ANKRD1-mediated ubiquitination of ACSL3 in H/R-treated HK-2 cells.A. Detection of endogenous ubiquitination levels of ACSL3 in H/R-treated HK-2 cells.B. Representative immunoblot images of ACSL3 in H/R-treated HK-2 cells after knockdown of the indicated genes.C. Renal IRI led to an increase in the endogenous binding of ANKRD1 and ACSL3 with TRIM25.D. High levels of TRIM25 were observed to augment endogenous ubiquitination of ACSL3 in H/R-stimulated HK-2 cells.Myc tagged TRIM25 was transfected into HK-2 cells, and endogenous ubiquitination levels of ACSL3 were detected by immunoprecipitation and western blot analysis.

Fig. S11 .
Fig. S11.Interfering with ANKRD1 levels does have minimal impact on the transcription of ACSL3 and TRIM25 in vivo and in vitro.Relative mRNA levels of ACSL3 and TRIM25 in vivo and in vitro after intervention with ANKRD1.ns.no significant.

Fig. S1 .Fig
Fig.S1.Altering ANKRD1 expression can not significantly impact P53/SLC7A11 levels in both in vitro and in vivo IRI models.